Project Summary The great promise of human microbiome research is that managing microbiome composition will prevent and cure disease. To this end, our long-term goals are to identify molecular mechanisms that drive bacterial community dynamics in nasal and skin microbiota and to uncover new antibiotics for controlling pathogen colonization and infection. Previous research shows that nasal microbiome composition and Staphylococcus aureus nasal colonization are strongly impacted by environmental factors, including other members of the microbiome. However, the molecular underpinnings of this are largely unknown. This proposal focuses on Propionibacterium species that colonize the skin and nasal passages of most adult humans. Our overarching hypothesis is that cutaneous Propionibacterium species produce antibiotics that control colonization by and proliferation of S. aureus and pathogenic Streptococcus species. Our preliminary data indicate that cutaneous Propionibacterium encode a distinct set of biosynthetic gene clusters (BGCs) predicted to produce antibiotics. We recently purified and structurally characterized one we call propimycin, a Propionibacterium-produced thiopeptide with potent activity against S. aureus in vitro. Our objectives are to identify more of these antibiotics and to define their role in shaping microbiome in vivo. Our rationale is that these Propionibacterium-produced compounds are likely to be important drivers of skin/nasal microbiome composition and, thus, key to developing new approaches to manage microbiome to promote health. To date, we have identified two Propionibacterium antibiotic BGCs specifically induced by coculture with S. aureus, propimycin and a nonribosomal peptide (NRP) that is widely distributed on human skin based on metagenomic data. We will use chemistry, bioinformatic, transcriptomic and bacterial genetics approaches to achieve the Specific Aims of this proposal: (1) determine the role of propimycin on human skin, focusing on skin follicles; (2A) purify and solve the structure of the NRP, a second candidate Propionibacterium antibiotic predicted to have anti-S. aureus activity; (2B) determine the mechanism of the NRP's increased transcription in response to Staphylococcus; and (3) identify additional candidate Propionibacterium antibiotic BGCs expressed in the context of the skin or nasal microbiome. Our strategy has four main innovations: (1) doing molecular experimental work in cutaneous Propionibacterium, which are recalcitrant to genetic manipulation; (2) using sebaceous plugs from human skin follicles to develop ex vivo assays to assess the impact of Propionibacterium antibiotics on S. aureus; (3) coupling a genetic screen in a heterologous system with next-gen-sequencing-based identification of promoters bound by candidate Propionibacterium acnes transcriptional regulators; and (4) using interactions between commensal Propionibacterium and the pathogens S. aureus and S. pneumoniae to uncover molecular mechanisms governing microbiome community dynamics. The significance will be to uncover new antibiotics produced by Propionibacterium and define their role in structuring skin/nasal microbial communities.